Affiliation:
1. From the Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK.
Abstract
Objectives—
To define the mechanism of p47
phox
phosphorylation in regulating endothelial cell response to tumor necrosis factor-α (TNFα) stimulation.
Methods and Results—
We replaced 11 serines (303-4, 310, 315, 320, 328, 345, 348, 359, 370, and 379) with alanines and investigated their effects on TNFα (100 U/mL, 30 minutes)–induced acute O
2
.−
production and mitogen-activated protein kinase phosphorylation in endothelial cells. Seven constructs, S303-4A (double), S310A, S315A, S328A, S345A, S370A, and S379A, significantly reduced the O
2
.−
production, and 4 of them (S328A, S345A, S370A, and S379A) also inhibited TNFα-induced extracellular-signal–regulated kinase (ERK) 1/2 phosphorylation. Blocking the phosphorylation of S303-4 and S379 inhibited most effectively TNFα-induced O
2
.−
production. However, phosphorylation of S303-4 was not required for TNFα-induced p47
phox
membrane translocation and binding to TNF receptor–associated factor 4, ERK1/2 activation, and subsequent vascular cell adhesion molecule-1 expression. Knockout of p47
phox
or knockdown of TNF receptor–associated factor 4 using siRNA abolished TNFα-induced ERK1/2 phosphorylation, and inhibition of ERK1/2 activation significantly reduced the TNFα-induced vascular cell adhesion molecule-1 expression.
Conclusion—
Phosphorylation of p47
phox
at different serine sites plays distinct roles in endothelial cell response to TNFα stimulation. Double serine (S303-4) phosphorylation is crucial for acute O
2
.−
production, but is not involved in TNFα signaling through TNF receptor–associated factor 4 and ERK1/2. p47
phox
requires serine phosphorylation at distinct sites to support specific signaling events in response to TNFα.
Publisher
Ovid Technologies (Wolters Kluwer Health)
Subject
Cardiology and Cardiovascular Medicine
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